Two novel atypical PKC inhibitors; ACPD and DNDA effectively mitigate cell proliferation and epithelial to mesenchymal transition of metastatic melanoma while inducing apoptosis

Atypical protein kinase Cs (aPKC) are involved in cell cycle progression, tumorigenesis, cell survival and migration in many cancers. We believe that aPKCs play an important role in cell motility of melanoma by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). We have investigated the effects of two novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD) and 3,4-diaminonaphthalene-2,7-disulfonic acid (DNDA) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular docking data suggested that both inhibitors specifically bind to protein kinase C-zeta (PKC-zeta) and PKC-iota (PKC-iota) and kinase activity assays were carried out to confirm these observations. Both inhibitors decreased the levels of total and phosphorylated PKC-zeta and PKC-iota. Increased levels of E-cadherin, RhoA, PTEN and decreased levels of phosphorylated vimentin, total vimentin, CD44, beta-catenin and phosphorylated AKT in inhibitor treated cells. This suggests that inhibition of both PKC-zeta and PKC-iota using ACPD and DNDA downregulates EMT and induces apoptosis in melanoma cells. We also carried out PKC-iota and PKC-zeta directed siRNA treatments to prove the above observations. Immunoprecipitation data suggested an association between PKC-iota and vimentin and PKC-iota siRNA treatments confirmed that PKC-iota activates vimentin by phosphorylation. These results further suggested that PKC-iota is involved in signaling pathways which upregulate EMT and which can be effectively suppressed using ACPD and DNDA. Our results summarize that melanoma cells proliferate via aPKC/AKT/NF-kappa B medi-ated pathway while inducing the EMT via PKC-iota/ Par6/RhoA pathway. Overall, results show that aPKCs are essential for melanoma progression and metastasis, suggesting that ACPD and DNDA can be effectively used as potential therapeutic drugs for melanoma by inhibiting aPKCs.